Angiogenesis is the fundamental process by which new blood vessels are formed. The process involves the migration of vascular endothelial cells into tissue followed by the condensation of such endothelial cells into vessels. Angiogenesis may be induced by an exogenous angiogenic agent or may be the result of a natural condition. The process is essential to a variety of normal body activities such as reproduction, development and wound repair. Although the process is not completely understood, it involves a complex interplay of molecules that stimulate and molecules that inhibit the growth and migration of endothelial cells, the primary cells of the capillary blood vessels. Under normal conditions, these molecules appear to maintain the microvasculature in a quiescent state (i.e., without capillary growth) for prolonged periods which can last for several years or even decades. The turnover time for an endothelial cell is about one thousand days. However, under appropriate conditions (e.g., during wound repair), these same cells can undergo rapid proliferation and turnover within a much shorter period, and a turnover rate of five days is typical under these circumstances. (Folkman and Shing, 1989, J. Biol. Chem. 267(16):10931-10934; Folkman and Klagsbrun, 1987, Science 235:442-447).
Although angiogenesis is a highly regulated process under normal conditions, many diseases (characterized as xe2x80x9cangiogenic diseasesxe2x80x9d) are driven by persistent unregulated angiogenesis. In such disease states, unregulated angiogenesis can either cause a particular disease directly or exacerbate an existing pathological condition. For example, ocular neovascularization has been implicated as the most common cause of blindness and underlies the pathology of approximately twenty diseases of the eye. In certain previously existing conditions such as arthritis, newly formed capillary blood vessels invade the joints and destroy cartilage. In diabetes, new capillaries formed in the retina invade the vitreous humor and bleed, causing blindness.
Both the growth and metastasis of solid tumors are also angiogenesis-dependent (Folkman, 1986, J. Cancer Res. 46:467-473; Folkman, 1989, J. Nat. Cancer Inst. 82:4-6; Folkman et al. 1995, xe2x80x9cTumor Angiogenesis,xe2x80x9d Chapter 10, pp. 206-32, in The Molecular Basis of Cancer, Mendelsohn et al., eds. (W. B. Saunders). It has been shown, for example, that tumors which enlarge to greater than about 2 mm in diameter must obtain their own blood supply and do so by inducing the growth of new capillary blood vessels. After these new blood vessels become embedded in the tumor, they provide nutrients and growth factors essential for tumor growth as well as a means for tumor cells to enter the circulation and metastasize to distant sites, such as liver, lung or bone (Weidner 1991, New Eng. J. Med. 324(1):1-8). When used as drugs in tumor-bearing animals, natural inhibitors of angiogenesis can prevent the growth of small tumors (O""Reilly et al., 1994, Cell 79:315-328). Indeed, in some protocols, the application of such inhibitors leads to tumor regression and dormancy even after cessation of treatment (O""Reilly et al., 1997, Cell 88:277-285). Moreover, supplying inhibitors of angiogenesis to certain tumors can potentiate their response to other therapeutic regimens (e.g., chemotherapy) (see, e.g., Teischer et al., 1994, Int. J. Cancer 57:920-925).
Although several angiogenesis inhibitors are currently under development for use in treating angiogenic diseases (Gasparini, 1996, Eur. J. Cancer 32A(14):2379-2385), there are disadvantages associated with these proposed inhibitory compounds. For example, suramin is a potent angiogenesis inhibitor, but, at doses required to reach antitumor activity, causes severe systemic toxicity in humans. Other compounds, such as retinoids, interferons and antiestrogens appear safe for human use but have only a weak anti-angiogenic effect. Still other compounds may be difficult or costly to make. In addition, the simultaneous administration of several different inhibitors of angiogenesis may be needed for truly effective treatment.
There remains, therefore, a long felt need for the development of new methods and compositions for inhibiting angiogenesis. The present invention satisfies these needs.
The invention includes a method of inhibiting angiogenesis within a tissue. The method comprises providing exogenous PEDF to endothelial cells associated with the tissue under conditions sufficient for the PEDF to inhibit angiogenesis within the tissue.
In several embodiments, the tissue is eye tissue, is skin tissue, a tumor, a tissue within a joint, bone marrow, nasal epithelium, prostate or ovarian or endometrial tissue.
In one aspect, the method further comprises supplying another antiangiogenic factor to the cells in conjunction with PEDF.
In yet another aspect, the PEDF is provided to the cells by exposing a composition comprising PEDF polypeptide to the cells.
In a further aspect, the PEDF is provided to the cells by transferring to the cells a vector, the vector comprising an isolated nucleic acid encoding PEDF, whereby the PEDF is expressed in and secreted from the cells.
In one embodiment, the isolated nucleic encoding PEDF comprises SEQ ID NO:2, and in another embodiment, the isolated nucleic acid encoding PEDF encodes a biologically active fragment of PEDF. Preferably, the biologically active fragment of PEDF is contained within the amino acid sequence of SEQ ID NO:1. More preferably, the biologically active fragment of PEDF comprises from amino acid 44 and amino acid 121 of SEQ ID NO:1; and even more preferably, the biologically active fragment of PEDF comprises amino acids 44-77 of SEQ ID NO:1.
In other preferred embodiments, the PEDF comprises SEQ ID NO:1 or a biologically active fragment of SEQ ID NO:1, wherein, in other preferred embodiments, the biologically active fragment of PEDF comprises from amino acids 44 to amino acids 121 of SEQ ID NO:1, or preferably comprising amino acids 44-77 of SEQ ID NO:1.
In one embodiment, the PEDF is provided to the endothelial cells by transfecting into a population of other cells a vector, the vector comprising an isolated nucleic acid encoding PEDF, whereby the PEDF is expressed in and secreted from the other cells, and transferring the population of the other cells so transfected to a site where PEDF so secreted is cable of contacting the endothelial cells.
In one embodiment, the isolated nucleic acid is SEQ ID NO:2, or in another embodiment, the isolated nucleic acid is a biologically active fragment of PEDF. Preferably, the biologically active fragment of PEDF is encoded by a fragment of SEQ ID NO:2.
In another embodiment, transfection of the isolated nucleic acid into the population of other cells results in expression of PEDF from non-integrated or stably integrated DNA in the other cells.
In other aspects of the invention, the PEDF is supplied to the cells via the systemic circulation, or via topical administration.
The invention also includes a method of inhibiting endothelial cell migration. The method comprises providing exogenous PEDF to the cells under conditions sufficient for the PEDF to inhibit endothelial cell migration.
The invention further includes a method of stimulating the growth of hair in a mammal. This method comprises providing exogenous PEDF to cells associated with the skin of the mammal under conditions sufficient for the PEDF to stimulate the growth of hair in the mammal.
Also included is a method for inhibiting the growth of a tumor. This method comprises providing exogenous PEDF to endothelial cells associated with the tumor under conditions sufficient for the PEDF to inhibit the migration of the endothelial cells within and to the tumor such that the growth of the tumor is inhibited.
In one embodiment, the method further comprises supplying another antiangiogenic factor to the cells in conjunction with PEDF.
In another embodiment, the PEDF is provided to the cells by exposing a composition comprising PEDF polypeptide to the cells.
In yet another embodiment, the PEDF is provided to the cells by transferring to the cells a vector, the vector comprising an isolated nucleic acid encoding PEDF, whereby the PEDF is expressed in and secreted from the cells.
In another embodiment, the PEDF is provided to the endothelial cells by transfecting into a population of other cells a vector, the vector comprising an isolated nucleic acid encoding PEDF, whereby the PEDF is expressed in and secreted from the other cells, and transferring the population of the other cells so transfected to a site where PEDF so secreted is capable of contacting the endothelial cells.
In several embodiments, the PEDF is supplied to the cells via the systemic circulation or via topical administration.
The invention further includes a pharmacological composition comprising a source of PEDF and a suitable diluent. In one aspect, the source of PEDF is PEDF polypeptide. In another aspect, the source of PEDF is a vector comprising an isolated nucleic acid encoding PEDF.
Further included in the invention is a method of determining the severity of a tumor by assaying for the presence of PEDF within the tumor, wherein the absence of PEDF within the tumor indicates an advanced state and the presence of PEDF within the tumor indicates an early state of the tumor.
In addition, the invention includes a method of inducing differentiation of a neuroblastoma cell. The method comprises administering PEDF to the cell, thereby inducing differentiation of the cell.
The invention also includes a method of slowing the growth of a neuroblastoma cell. The method comprises administering PEDF to the cell, thereby slowing the growth of the cell.
The invention further includes a method of treating ischemic retinopathy in a mammal. The method comprises providing exogenous PEDF to endothelial cells associated with the eye of the mammal under conditions sufficient for the PEDF to inhibit angiogenesis in the eye, thereby treating the ischemic retinopathy.
Also included is a method of inhibiting angiogenesis within a tissue in a mammal. The method comprises providing exogenous PEDF systemically to the mammal, under conditions sufficient for the PEDF to inhibit angiogenesis within the tissue.
In one embodiment, the tissue is selected from the group consisting of eye tissue, skin tissue, a tumor, a tissue within a joint, bone marrow, nasal epithelium, prostate, ovarian and endometrial tissue. Preferably, the tissue is eye tissue.
Also preferably, the mammal is selected from the group consisting of a mammal that has ischemic retinopathy, a mammal that is at risk for developing ischemic retinopathy, a mammal that has macular degeneration and a mammal that is at risk for developing macular degeneration.
In another embodiment, the method further comprises supplying another antiangiogenic factor to the cells in conjunction with PEDF.
Also included is a method of upregulating the expression of PEDF in a cell in a tissue. This method comprises inducing hyperoxia in the tissue, thereby upregulating the expression of PEDF in the cell.
The invention further includes a method of treating macular degeneration in a mammal. The method comprises providing exogenous PEDF to endothelial cells associated with the eye of the mammal under conditions sufficient for said PEDF to inhibit angiogenesis in the eye, thereby treating the macular degeneration.
Also included is a method of treating a benign neoplasia in a mammal, the method comprising administering PEDF to the mammal, thereby treating the benign neoplasia. Preferably, the benign neoplasia is a nasal polyp, more preferably, in a human having cystic fibrosis. Also preferably, the benign neoplasia is in the prostate gland.
The invention further includes a method of inhibiting angiogenesis within a tissue. The method comprising providing exogenous PEDF to endothelial cells associated with the tissue in conjunction with at least one other treatment selected from the group consisting of radiation, chemotherapy, the use of at least one biological response modifier, laser treatment, under conditions sufficient for the PEDF to inhibit angiogenesis within the tissue.